The completed sequencing of all major genomes provided genome-wide insight into gene sequence and structure. However, our ability to functionally analyze such large gene sets is still limited. To develop novel technologies for more efficient functional gene annotation, we propose a multidisciplinary approach, using T cell receptor (TCR) signaling as an example of a physiologically and pathologically important cellular process that is still incompletely understood, but where available data enable integrated large-scale analysis to maximize novelty and in vivo relevance. If successful, the broad applicability of our "systems" approach to many areas of biology promises to accelerate generation of biological knowledge at large through its adoption by others, profoundly impacting large scientific and patient communities. As critical components of the adaptive immune system, T cells are key mediators of our defense against pathogens, cancer cells and, undesired, of transplant rejection. Impaired T cell function can result in immunodeficiency, promoting infections or cancer. T cell hyperactivity or deregulation is a main cause for allergies, autoimmune or neurodegenerative diseases. These serious disorders contribute to individual disability, loss of quality of life, inability to self-sustain, premature death and high economic burden for society and health care system. By identifying novel genes mediating T cell activation and analyzing their molecular functions, our proposed studies will thus promote our understanding of T cell function and malfunction in disease, and of signal transduction in general. By focusing on druggable genes whose functions can be modulated through small molecule or biological therapeutics, we aim to accelerate the development of new, improved and more cost-effective therapies for severe immune disorders or cancer that affect large patient populations. To devise and validate our innovative, integrated, multidisciplinary approach for gene discovery and analysis, we will first identify novel TCR signaling genes in a high-throughput RNA interference screen in T cells of an arrayed shRNA library that targets 5,000 druggable genes. We will use lentiviral shRNA delivery to overcome the challenges of nonadherent growth and relative T cell resistance to conventional robotic siRNA delivery methods, and novel screen designs and analysis strategies to maximize sensitivity and minimize false positive rates. We will next combine gene expression profiling, phosphotyrosine-proteomics, haplotype association mapping, quantitative trait locus (QTL), genome-wide associtation mapping and gene expression QTL data with gene/protein network and pathway analyses to prioritize those hits which combine highest novelty with maximal in vivo relevance for detailed follow-up.